Process for producing nocardicin A

ABSTRACT

A new species of Nocardiopsis, designated Nocardiopsis atra Huang sp. nov. ATCC 31511, when aerobically propagated in an aqueous nutrient medium, produces nocardicin A in good yield.

BACKGROUND OF THE INVENTION

Among the best known and widely used class of antibacterial agents are the so-called β-lactam antibiotics. These compounds are characterized in that they have a nucleus consisting of a 2-azetidinone (β-lactam) ring. When the β-lactam is fused to a thiazolidine ring, the compounds are usually referred to generically as penicillins. When the β-lactam is fused to a dihydrothiazine ring, the compounds are referred to as cephalosporins.

The β-lactam antibiotics are synthesized, for no apparent reason, by only a few microorganisms. All of the organisms recognized to produce β-lactam antibiotics are filamentous microorganisms, but not all of these microorganisms are taxonomically related. Some are fungi (eukaryotes), whereas others are streptomycetes (prokaryotes). Thienamycin, U.S. Pat. No. 3,950,357, is an interesting new β-lactam antibiotic produced by a species of Streptomyces.

Nocardicin A, U.S. Pat. No. 3,923,977, is a novel β-lactam antibiotic produced by Nocardia uniformis var. tsuyamanensis ATCC 21806. Nocardicin A has an unusual β-lactam moiety which is uniquely different from all other β-lactam antibiotics in not being fused to another ring as shown in the following structure: ##STR1##

The unique structure of nocardicin A may account for its relative stability toward a variety of β-lactamases as well as for its negligible immunological cross-reactivity with penicillins and cephalosporins.

Unfortunately, the outstanding in vitro and in vivo antimicrobial properties of the penicillins and caphalosporins are not shared by nocardicin A. However, the antibiotic's nucleus, 3-aminonocardicinic acid, of the following structure has been prepared, J. Am. Chem. Soc. 100, 3933 (1978): ##STR2##

The present invention is concerned with the discovery that nocardicin A is produced by a new species of Nocardiopsis designated Nocardiopsis atra Huang sp. nov. ATCC 31511. The microorganism with its potential for cultural and mutational development offers promise of the production of large amounts of nocardicin A, subsequent cleavage to 3-aminonocardicinic acid and the synthesis of a variety of clinically useful semisynthetic nocardicins paralleling those of the semisynthetic penicillins and cephalosporins.

SUMMARY OF THE INVENTION

Nocardicin A, previously reported β-lactam antibiotic, is produced by the aerobic propagation in aqueous nutrient media of a new species of Nocardiopsis designated Nocardiopsis atra Huang sp. nov. ATCC 31511.

DETAILED DESCRIPTION OF THE INVENTION

The microorganism useful for the production of nocardicin A was isolated from a coil sample from Nomozaki, Japan. This culture, designated Nocardiopsis atra Huang sp. nov., has been deposited in The American Type Culture Collection, Rockville, Md. as the type culture under their accession number ATCC 31511. The permanency of the deposit of this culture at The American Type Culture Collection at Rockville, Md. and ready accessibility thereto by the public are afforded throughout the effective life of the patent in the event the patent is granted. Access to the culture is available during pendency of the application under 37 CFR 1.14 and 35 USC 112. All restrictions on the availability to the public of the culture deposited will be irrevocably removed upon granting of the patent.

The culture was planted from a slant into liquid ATCC No. 172 medium and grown for 4 days at 28° C. on a shaker. It was then removed from the shaker, centrifuged for 20 minutes, washed three times with sterile distilled water and planted on media commonly used for identification of members of the Actinomycetales.

The inoculated plates were incubated at 28° C. and records of results were made after suitable incubation time with most of the final results recorded at 14 days. The colors were described in common terminology, but exact color was determined by comparison with color chips from the Color Harmony Manual, fourth edition.

The methods of whole-cell and sugar analyses are those described by Becker, B. et al., Appl. Microbiol., 12, 421-423 (1964) and by Lechevalier, M.P., J. Lab. Clin. Med., 71, 934-944 (1968). Mycolate analyses were performed by the method of Lechevalier, M.P. et al., J. Bacteriol., 105, 313-318 (1971).

The results of chemical tests of whole-cell and cell wall indicated the new culture belonged in the genus Nocardiopsis. For comparison purposes, therefore, culture studies included Nocardia uniformis subsp. tsuyamanesis ATCC 21806 and Nocardiopsis dassonvillei ATCC 23218.

Identification media used for the characterization of the culture and references for their composition are as follows:

1. Tryptone Yeast Extract Broth (ISP #1 medium, Difco).

2. Yeast Extract-Malt Extract Agar (ISP #2 medium, Difco).

3. Oatmeal Agar (ISP #3 medium, Difco).

4. Inorganic Salts--Starch Agar (ISP #4 medium, Difco).

5. Glycerol--Asparagine Agar (ISP #5 medium, Difco).

6. Glycerol--Asparagine Agar (prepared in our lab).

7. Peptone--Yeast Extract Iron Agar (#6 medium, Difco).

8. Gelatin Agar--R. E. Gordon and J. M. Mihm, Jr. Bact. 73: 15-27, 1957.

9. Starch Agar--Ibid.

10. Organic Nitrate Broth--Ibid.

11. Dextrose Nitrate Broth--S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1. p. 328, 1961, and 3 g dextrose substituted for 30 g sucrose and agar omitted.

12. Potato Carrot Agar--M. P. Lechevalier, Jr. Lab. and Clinical Med. 71: 934-944, 1968 but use only 30 g potatoes, 2.5 g carrots and 20 g agar.

13. 2% Tap Water Agar

14. Czapek-Sucrose Agar--S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1, p. 328, 1961.

15. Glucose Asparagine Agar--Ibid, medium no. 2, p. 328.

16. Glucose-Yeast Extract Agar--Ibid, medium no. 29, p. 331

17. Emerson's Agar--Ibid, medium no. 28, p. 331.

18. Nutrient Agar--Ibid, medium no. 14, p. 330.

19. Bennet's Agar--Ibid, medium no. 30, p. 331.

20. Gordon and Smith' Tyrosine Agar R. E. Gordon and M. M. Smith, Jr. Bact. 69: 147-150, 1955.

21. Casein Agar--Ibid.

22. Calcium Malate Agar--S. A. Waksman, Bact. Rev. 21: 1-29, 1957.

23. Gauze's #1 Mineral Agar--G. F. Gauze et al. Problems in the Classification of Antagonistic Actinomycetes. English Ed., p. 13, 1957.

24. Gauze's #2 Organic Agar--Ibid.

25. Skim Milk--Difco

26. Cellulose utilization--

(a) H. L. Jensen, Proc. Linn. Soc. N.S.W. 55: 231-248, 1930.

(b) M. Levine and H. W. Schoenlein, A Compilation of Culture Media, Medium 2511, 1930.

27. Utilization of Organic Acids--R. E. Gordon et al., Int. Jr. Syst. Bact. 24: 54-63, 1974.

28. Carbohydrate Utilization and Acid Production from Carbohydrates--Ibid.

29. Hydrolysis of Hippurate and Esculin--Ibid.

30. Decomposition of Adenine, Hypoxanthine, Xanthine, and Urea--Ibid.

31. Yeast Dextrose Agar for Studies of Temperature and Survival at 50° C.--Ibid.

32. Resistance to Lysozyme--Ibid.

33. Glucose Broth--Ibid.

    __________________________________________________________________________                            Nocardia                                                              Nocardiopsis                                                                            uniformis subsp.                                                                         Nocardiopsis                                          Cultural                                                                             atra     tsuyamanensis                                                                            dassonvillei                                  Agar    Character*                                                                           ATCC 31511                                                                              ATCC 21806                                                                               ATCC 23218                                    __________________________________________________________________________     Yeast Extract-                                                                         G     Poor to moderate                                                                        Good      Excellent                                     Malt Extract                                                                           SC    Brown to greyish                                                                        Orange    White with pale                               Agar          brown    (4 ia to 4 lc)                                                                           orange tint                                                 (4 lg to 4 li)     (3 ea)                                                T     Thin, smooth to                                                                         Thin to slightly                                                                         Wrinkled with a                                             slightly Sc                                                                             raised, wrinkled                                                                         fluffy edge,                                          AM    --       --        White                                                 RC    Same as SC                                                                              Same as SC                                                                               Yellowish orange                                                               (3 ia to 3 lc)                                        SP    --       --        Pale yellowish                                Oatmeal Agar                                                                           G     Moderate Moderate  Moderate                                              SC    Dull white                                                                              Pale brownish                                                                            Colorless to                                                         (3 gc)    white                                                 T     Thin, smooth,                                                                           Submerged, smooth                                                                        Thin, smooth with                                           with a few small                                                                        with spreading                                                                           spreading edge                                              white dots                                                                              edge                                                            AM    None to scant                                                                           --        White, sparse                                         RC    Same as SC                                                                              Same as SC                                                                               Same as SC                                            SP    Pale greenish                                                                           --        --                                            Inorganic                                                                              G     Moderate Moderate to good                                                                         Moderate                                      Salts-Starch                                                                           SC    Pale grey to                                                                            Yellowish orange                                                                         White to pale                                 Agar          greyish (near                                                                           (3 ea, 3 ia to                                                                           yellowish                                                   grey series,                                                                            3 la)     (2 ga)                                                      2 dc to 2 fe)                                                                  with white tint                                                          T     Thin, smooth                                                                            Thin to slightly                                                                         Thin to slightly                                                     raised    raised                                                                         smooth to                                                                      slightly                                                                       wrinkled                                              AM    White    --        White                                                 RC    Same as surface                                                                         Same as surface                                                                          Same as surface                                       SP    Pale greyish green                                                                      --        --                                                          (1 gc)                                                           Glycerol                                                                               G     Poor to moderate                                                                        Poor to moderate                                                                         Poor                                          Asparagine                                                                             SC    Colorless to cream                                                                      Colorless to pale                                                                        Colorless to                                  Agar                   yellowish brown                                                                          pale cream                                    (dehydrated            (near 2 ea)                                             powder from                                                                            T     Thin, appearing as                                                                      Submerged, smooth                                                                        Thin, smooth,                                 Difco)        confluent smear    with a fluffy                                               or very small      edge                                                        isolated dots                                                            AM    --       --        --                                                    RC    Same as surface                                                                         Same as surface                                                                          Same as surface                                       SP    --       --        --                                            Glycerol                                                                               G     Good     Moderate  Moderate to good                              Asparagine                                                                             SC    Grey, dark grey to                                                                      Orange    White to cream                                Agar          black (near grey)                                                                       (3 ga to 3 ia)                                                                           (1 1/2 ca)                                    (Prepared)    series, 3 ih to                                                                3 ml)                                                                    T     Raised, slightly                                                                        Thin, smooth, with                                                                       Raised, rough-                                              wrinkled a spreading                                                                              ened to                                                              edge      wrinkled                                              AM    --       --        White                                                 RC    Brown (4 pg to                                                                          Same as surface                                                                          Pale yellowish                                              4 pi) to black     to pale yel-                                                                   lowish green                                                                   (1 1/2 ea to                                                                   1 1/2 gc)                                             SP    Greyish orange                                                                          Very pale Pale yellowish                                              (near 3 gc) to                                                                          orange                                                                black                                                            Gelatin Agar                                                                           G     Good     Good      Good                                                  SC    Grey to black                                                                           Orange (3 ia)                                                                            Cream to pale                                               (near grey         yellowish                                                   series, 3 fe)      (2 ca to 2 ga)                                        T     Raised, wrinkled                                                                        Thin, smooth but                                                                         Thin, smooth but                                                     slightly wrin-                                                                           slightly wrin-                                                       kled near the                                                                            kled near the                                                        edge      end of streak                                         AM    --       --        White                                                 RC    Same as surface                                                                         Same as surface                                                                          Yellowish (2 ia)                                      SP    Greyish  --        --                                            Starch Agar                                                                            G     Good     Good      Good                                                  SC    Greyish to black                                                                        Orange (near 4 ia                                                                        Yellowish brown                                             (near grey                                                                              or 4 la)  (near 2 lc)                                                 series 2 ih to     but white                                                   7 ml               near edge                                             T     Raised, roughened                                                                       Slightly raised,                                                                         Slightly raised,                                                     wrinkled  wrinkled                                              AM    --       --        White                                                 RC    Same as surface                                                                         Same as surface                                                                          Same as surface                                       SP    Greyish  --        --                                            Potato Carrot                                                                          G     Moderate Poor to moderate                                                                         Poor to moderate                              Agar    SC    Cream (1 1/2                                                                            Colorless to pale                                                                        Colorless, white                                            ca)      brown (2 ca)                                                                             to pale brown                                                                  (2 ca)                                                T     Thin, smooth,                                                                           Submerged, thin,                                                                         Thin, smooth,                                               with some small                                                                         smooth, with a                                                                           with a fluffy,                                              slightly raised                                                                         spreading edge                                                                           spreading edge                                              dots                                                                     AM    None to sparse                                                                          --        White                                                 RC    Same as surface                                                                         Same as surface                                                                          Same as surface                                       SP    --       --        --                                            Tap Water                                                                              G     Poor to moderate                                                                        Poor      Poor                                          Agar    SC    Colorless to white                                                                      Colorless Colorless                                             T     Thin, smooth, with                                                                      Submerged, smooth,                                                                       Thin, smooth                                                a few white dots                                                                        with a spread-                                                                 ing edge                                                        AM    None to sparse                                                                          --        --                                                    RC    Same as surface                                                                         Same as surface                                                                          Same as surface                                       SP    --       --        --                                            Czapek-Sucrose                                                                         G     Moderate Moderate  Moderate                                      Agar    SC    Pale greyish green                                                                      Yellowish to                                                                             Cream (1 1/2 ca)                                            (1 1/2 ec) with                                                                         yellowish with very pale                                              some small black                                                                        orange (1 1/2 ea                                                                         greenish tint                                               dots, greyish                                                                           to 2 lc)                                                              near the end of                                                                streak (near grey                                                              series, 2 fe)                                                            T     Thin, smooth                                                                            Submerged, smooth                                                                        Slightly raised                                                      with a spread-                                                                           and roughened                                                        ing edge                                                        AM    --       --        White, sparse                                         RC    Same as surface                                                                         Same as surface                                                                          Same as surface                                       SP    Pale greenish                                                                           --        Pale greyish                                                (1 1/2 gc)                                                       Glucose G     Moderate Moderate  Moderate                                      Asparagine                                                                             SC    Pale brown (3 gc)                                                                       Yellowish orange                                                                         Cream (1 1/2 ca)                              Agar          to black (3 ga to 3 la)                                                  T     Slightly raised,                                                                        Thin, smooth, with                                                                       Thin to slightly                                            smooth, with                                                                            a few small                                                                              raised appear-                                              small raised                                                                            raised dots                                                                              ing as small                                                black dots         isolated dots,                                                                 slightly                                                                       wrinkled                                              AM    --       --        --                                                    RC    Brown to black                                                                          Same as surface                                                                          Same as surface                                       SP    Pale yellowish                                                                          --        --                                                          brown                                                            Glucose Yeast                                                                          G     Good     Good      Good                                          Extract Agar                                                                           SC    Black (near grey                                                                        Orange    White to cream                                              series, 3 po)                                                                           (near 4 ia)                                                                              (2 ca)                                                T     Raised, wrinkled                                                                        Slightly raised,                                                                         Raised, strongly                                                     wrinkled  wrinkled                                              AM    --       --        White                                                 RC    Black    Same as surface                                                                          Yellowish brown                                                                (near 2 ic)                                           SP    Black    --        Brown (3 ic)                                  Emerson's                                                                              G     Good to excellent                                                                       Good      Good                                          Agar    SC    Black (near grey                                                                        Orange    White, cream to                                             series, 3 ml)                                                                           (4 ga to 4 ia)                                                                           brown (2 ca to                                                                 3 le)                                                 T     Raised, wrinkled                                                                        Slightly raised,                                                                         Slightly raised,                                                     wrinkled, but                                                                            roughened to                                                         smooth near                                                                              wrinkled, with                                                       the edge  a fluffy,                                                                      spreading edge                                        AM    --       --        White                                                 RC    Black    Same as surface                                                                          Brown                                                 SP    Black    Yellowish brown                                                                          Brown (4 ne)                                                         (3 lc)                                                  Nutrient Agar                                                                          G     Moderate to poor                                                                        Moderate  Moderate                                              SC    Pale grey to                                                                            Pale orange                                                                              Cream (2 ca)                                                grey (near grey                                                                         (between 3 ea                                                         series, 2 dc,                                                                           4 ea)                                                                 2 ih to 2 ml)                                                            T     Thin, smooth, with                                                                      Thin, smooth                                                                             Thin, smooth or                                             raised black       appearing as                                                dots               small isolated                                                                 dots, with a                                                                   spreading edge                                        AM    --       --        White, sparse                                         RC    Same as surface                                                                         Same as surface                                                                          Cream to pale                                                                  yellowish                                             SP    Pale greyish                                                                            --        --                                            Bennett's Agar                                                                         G     Good to excellent                                                                       Moderate to good                                                                         Good                                                  SC    Black (near grey                                                                        Yellowish orange                                                                         Cream to yellow-                                            series, 3 ml)                                                                           (3 ga to 3 la)                                                                           ish (2 ca to                                                                   2 ea) with a                                                                   white edge                                            T     Raised, wrinkled,                                                                       Thin, smooth to                                                                          Moderately                                                           slightly wrin-                                                                           raised, wrin-                                                        kled      kled, with a                                                                   fluffy edge                                           AM    --       --        White                                                 RC    Black    Same as surface                                                                          Same as surface                                       SP    Black    --        --                                            Gordon and                                                                             G     Moderate Moderate to good                                                                         Moderate                                      Smith'  SC    Grey to dark grey                                                                       Dark orange                                                                              Dirty white                                   Tyrosine      (near grey                                                                              (near 4 lc)                                             Agar          series, 2 ih                                                                   to 2 ml)                                                                 T     Thin to slightly                                                                        Thin, smooth                                                                             Thin, smooth or                                             raised, slightly   appearing as                                                roughened to       small isolated                                              granular           dots, with a                                                                   spreading edge                                        AM    --       --        White                                                 RC    Same as surface                                                                         Same as surface                                                                          Pale yellowish                                        SP    Greyish black                                                                           Brown (near 3 ic)                                                                        Brown (between                                                                 3 gc and 3 ic)                                Calcium G     Moderate Poor to moderate                                                                         Moderate                                      Malate  SC    Black    Yellowish to                                                                             Dirty white to                                Agar                   yellowish brown                                                                          cream                                                                (2 ga to 2 lc)                                                                           (2 ca)                                                T     Thin, smooth, with                                                                      Thin, smooth, with                                                                       Appearing as                                                some raised dots                                                                        a spreading sub-                                                                         isolated dots                                                        merged edge                                                     AM    --       --        White                                                 RC    Black    Same as surface                                                                          Yellowish brown                                                                (2 ic)                                                SP    Pale greenish                                                                           --        --                                            Gauze's #1                                                                             G     Moderate Moderate to good                                                                         Moderate to good                              Mineral Agar                                                                           SC    Pale greenish                                                                           Yellowish orange                                                                         White                                                       cream    (3 ea to 3 ia)                                                  T     Thin, with small                                                                        Thin, smooth, with                                                                       Slightly raised                                             white to greyish                                                                        a spreading edge                                                                         and wrinkled                                                dots                                                                     AM    White, sparse                                                                           --        White                                                 RC    Same as surface                                                                         Same as surface                                                                          Greyish brown                                                                  (2 ie)                                                SP    Pale greenish (1 ca)                                                                    --        Pale greyish                                                                   (betwen 2 ec                                                                   and 2 ge)                                     Gauze's #2                                                                             G     Moderate to good                                                                        Good      Moderate to good                              Organic Agar                                                                           SC    Dark brown to                                                                           Orange (3 ga,                                                                            Cream                                                       greyish (3 ni,                                                                          3 ia to 3 gc)                                                         near grey series                                                               3 ih)                                                                    T     Raised, granular                                                                        Slightly raised,                                                                         Raised and                                                  to flaky wrinkled, with                                                                           wrinkled                                                             a moderately                                                                   spreading edge                                                  AM    --       --        --                                                    RC    Dark brown to                                                                           Same as surface                                                                          Same as surface                                             black                                                                    SP    Greyish brown                                                                           Pale yellowish                                                                           Pale yellowish                                                       orange                                                  __________________________________________________________________________      *G-growth, SCsurface color, Ttexture, AMaerial mycelium, RCreverse color,      SPsoluble pigment, " means none.                                         

    ______________________________________                                                               Nocardia                                                 Biochemical and                                                                            N. atra   uniformis subsp.                                                                           Nocardiopsis                                 Physiological                                                                              ATCC      tsuyamanensis                                                                              dassonvillei                                 Properties  31511     ATCC 21806  ATCC 23218                                   ______________________________________                                         Gram stain  +         +           +                                            Acid-fastness                                                                              -         -           -                                            Decomposition of:                                                              Adenine     -         -           +                                            Calcium malate                                                                             +         +           +                                            Casein      +         +           +                                            Hypoxanthine                                                                               -         -           +                                            Tyrosine    +         -           -                                            Urea        +         +           +                                            Xanthine    -         -           +                                            Hydrolysis of:                                                                 Esculin     -         +           +                                            Hippurate   +         -           +                                            Starch      +         +           +                                            Growth at:                                                                     45° C.                                                                              -         -           -                                            37° C.                                                                              very poor good        good                                         28° C.                                                                              good      good        good                                         21° C.                                                                              very poor good        good                                         Survival at 50° C.                                                                  -         +           -                                            8 h                                                                            Melanin production                                                                         -         -           -                                            H.sub.2 S production                                                                       -         +           +                                            Gelatin liquefaction                                                                       +         +           +                                            Cellulose                                                                      decomposition                                                                              -         -           -                                            Growth in                                                                      cellulose broth:                                                               Jensen's    poor      good        poor                                         Levine &                                                                       Schoenlein's                                                                               -         poor        -                                            Skim milk:                                                                     Coagulation -         +           -                                            peptonization                                                                              -         +           -                                            Acid from and                                                                  (utilization of):                                                              Adonitol    + (+)     - (-)       - (-)                                        Arabinose   + (+)     + (±)    + (+)                                        Cellobiose  + (+)     + (+)       + (+)                                        Dulcitol    - (-)     - (+)       - (-)                                        Erythritol  - (+)     - (±)    - (-)                                        Fructose    + (+)     + (+)       + (+)                                        Galactose   + (+)     + (+)       + (+)                                        Glucose     + (+)     + (+)       + (+)                                        Glycerol    + (+)     + (+)       + (+)                                        Inositol    + (+)     - (-)       - (-)                                        Lactose     + (+)     + (+)       - (±)                                     Maltose     + (+)     + (+)       + (+ )                                       Mannitol    + (+)     + (+)       + (+)                                        Mannose     + (+)     + (+)       + (+)                                        Melezitose  - (±)  - (±)    - (±)                                     Melibiose   + (+)     + (+)       - (-)                                        α-Methyl-d-                                                                          + (+)     - (-)       - (±)                                     glucoside                                                                      Raffinose   + (+)     + (+)       - (±)                                     Rhamnose    + (+)     + (+)       + (+)                                        Ribose      + (+)     - (-)       + (+)                                        Salicin     + (+)     + (+)       + (+)                                        Sorbitol    - (+)     - (±)    - (-)                                        Sorbose     - (±)  - (±)    - (-)                                        Starch      + (+)     + (+)       + (+)                                        Sucrose     + (+)     + (+)       + (+)                                        Trehalose   + (+)     + (+)       + (+)                                        Xylose      + (+)     + (+)       + (+)                                        Utilization of:                                                                Acetate     +         +           +                                            Benzoate    -         -           -                                            Citrate     +         -           +                                            Dextrin     -         -           -                                            Lactate     +         +           +                                            Malate      +         +           +                                            Mucate      -         -           -                                            Oxalate     -         -           -                                            Phenol      -         -           -                                            Propionate  -         -           -                                            Pyruvate    +         +           +                                            Succinate   +         +           +                                            Nitrite from                                                                   Nitrate:                                                                       Dextrose nitrate                                                                           -         +           +                                            broth                                                                          Organic nitrate                                                                            -         +           +                                            broth                                                                          Resistance to                                                                              -         +           -                                            lysozyme                                                                       ______________________________________                                          "+", positive; "-", negative; "±", doubtfully utilized.               

Morphological observations were made 1, 2, 3, 5, 7, 14 and 28 days after the inoculation of the new Nocardiopsis culture on Czapek sucrose agar. Substrate mycelium began to fragment into bacillary cells after 3 days of incubation; aerial mycelium appeared after 5 days of incubation; substrate and aerial mycelia fragmented after 14 days of incubation into rods of varying lengths which were smooth and measured 1.5-10 (or longer)×0.6-0.9 μm.

The cell wall contained meso-diaminopimelic acid but not diagnostic sugars. The cell wall contained no mycolates of any kind.

The new culture is characterized by gram-positive reaction, non-acid-fastness, black or greyish colonies, black or greyish colony reverse, black soluble pigment on some media and fragmentation of both aerial and substrate mycelia. These features plus the presence of mesodiaminopimelic acid in the cell wall but the absence of diagnostic sugars and mycolates place the new culture in the genus Nocardiopsis.

The biochemical and physiological properties distinguishing the new culture from Nocardia uniformis subsp. tsuyamanesis are the decomposition of tyrosine; hydrolysis of esculin and hippurate; growth at 21° and 37° C. survival at 50° C. for 8 hours; H₂ S production; growth in Jensen's cellulose broth; coagulation and peptonization of milk; nitrate reduction; resistance to lysozyme; acid production after utilization of adonitol, inositol, α-methyl-d-glucoside and ribose. The new culture differs from Nocardiopsis dassonvillei in hydrolysis of esculin; growth at 21° and 37° C.; H₂ S production, nitrate reduction; decomposition of adenine, hypoxanthine tyrosine and xanthine; acid production from adonitol, inositol, lactose, melibiose, α-methyl-d-glucoside and raffinose; and utilization of adonitol, erythritol, inositol, melibiose and sorbitol.

The new culture was considered to be a new species of Nocardiopsis and was designated as Nocardiopsis atra Huang sp. nov.

Cultivation of Nocrdiopsis atra Huang sp. nov. ATCC 31511 preferably takes place in aqueous nutrient media at a temperature of 24-36° C. and under submerged aerobic conditions with agitation. Nutrient media which are useful for such purposes include a source of assimilable carbon such as sugars, starches and glycerol; a source of organic nitrogen such as casein, enzymatic digest of casein, soybean meal, cottonseed meal, peanut meal, wheat gluten, soy flour, meat meal and fish meal. A source of growth substances such as grain solubles and yeast extract as well as salts such as sodium chloride and calcium carbonate and trace elements such as iron, zinc, cobalt and maganese may also be utilized with advantageous results. If excessive foaming is encountered during fermentation, antifoam agents such as vegetable oil or silicones may be added to the fermentation medium. Aeration of the medium in tanks for submerged growth is preferably maintained at the rate of about 1/2 to 2 volumes of free air per volume of broth per minute. Agitation may be maintained by means of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained through the transfer of the organism and throughout its growth.

Inoculum for the preparation of the antibiotic may be obtained by employing growth from a slant of the culture. The growth may be used to inoculate either shake flasks or inoculum tanks or the inoculum tanks may be seeded from the shake flasks. Growth in shaken flasks will generally have reached its maximum in 2 to 4 days whereas inoculum in submerged inoculum tanks will usually be at the most favorable period in 1.5-3 days. Substantial antibiotic activity is obtained in the final fermentor stage in approximately 2 to 5 days.

The process of antibiotic production is conveniently followed during fermentation by biological assay of the broth employing a sensitive strain of Comamonas terrigena ATTC 8461 or Micrococcus lutenus. Standard plate assay technique is employed in which the zone of inhibition surrounding a filter paper disc saturated with broth is used as a measure of antibiotic potency.

The antibiotic produced by Nocardiopsis atra Huang sp. nov. ATCC 31511 was recovered from the filtered fermentation broth and purified by a combination of carbon absorption and elution and column chromatography. The antibiotic was established to be identical with previously reported nocardicin A (U.S. Pat. No. 3,923,977) by comparison with the published physicochemical data and side by side comparisons with nocardicin A obtained from the fermentation broth of known producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806.

EXAMPLE 1

A sterile aqueous medium having the following composition is prepared:

    ______________________________________                                         Ingredient           Grams/liter                                               ______________________________________                                         Glucose              25                                                        Corn steep liquor    15                                                        Distillers' solubles 10                                                        Cottonseed meal      5                                                         Cobalt chloride      0.01                                                      Calcium carbonate    3                                                         ______________________________________                                    

Cells from a slant culture of Nocardiopsis atra ATCC 31511 are transferred to each of a number of 300 ml shake flasks each containing 40 ml of the above medium and shaken for 3 to 4 days at 28° C.

Aliquots sufficient to provide a 5% v/v inoculum are transferred to fermentors each containing two liters of the above described sterile medium. The temperature is maintained at 30° C. The broth is stirred at 200 r.p.m. and aerated at the rate of about one volume of air per volume of broth per minute. The fermentation is maintained until substantial antibiotic activity is obtained (3-4 days).

Nocardicin A may be recovered and separated by the methods described in U.S. Pat. No. 3,923,977.

EXAMPLE II

The method of Example I may be repeated with comparable results employing a fermentation medium of the following composition:

    ______________________________________                                         Ingredient           Grams/liter                                               ______________________________________                                         Glucose              30                                                        Corn steep liquor    6                                                         Glycine              3                                                         Cottonseed meal      6                                                         DL-methionine        2                                                         Calcium carbonate    5                                                         ______________________________________                                    

EXAMPLE III

The method of Example I may be repeated with comparable results employing a fermentation medium of the following composition:

    ______________________________________                                         Ingredient           Grams/liter                                               ______________________________________                                         Glucose              1                                                         Dextrin              24                                                        Yeast extract        5                                                         Beef extract         3                                                         Polypeptone          5                                                         Calcium carbonate    4                                                         ______________________________________                                     

We claim:
 1. A process for producing nocardicin A which comprises aerobically propagating Nocardiopsis atra Huang sp. nov. ATCC 31511 in an aqueous nutrient medium containing a source of assimilable carbon and a source of assimilable nitrogen until a substantial amount of nocardicin A is obtained. 